Protein-tyrosine kinases of the Src family are negatively regulated by two intramolecular interactions. The Src homology 2 (SH2) domain associates with the tyrosine-phosphorylated C-terminal tail, while the SH3 domain binds to a polyproline type II helix formed by the linker connecting the SH2 and kinase domains. By virtue of its unusually high affinity for SH3 domains, the Nef protein of HIV-1 provides a unique tool with which to probe the SH3 contribution to the regulatory mechanism. Recent work from our laboratory shows that SH3-mediated interaction of the Src family kinase Hck with Nef induces Hck activation and transformation of Rat-2 fibroblasts. In this application, the mechanism of Hck activation by Nef will be investigated with the following Specific Aims: 1) Determine whether activation of Hck by Nef affects the status of tail phosphorylation. Activation of Hck by Nef without a requirement for tail dephosphorylation will provide strong evidence that SH3 engagement alone is sufficient for Src family kinase activation in vivo. Such a result would suggest that interaction of Src kinases with substrates known to bind at the SH3 domain may induce kinase activation. 2) Create Hck mutants that mimic the effect of Nef SH3 binding by altering residues in the SH2-kinase linker region observed to make direct SH3 contacts in the crystal structure. If these contacts are essential for negative regulation, then mutations that disturb this interaction are predicted to activate Hck. 3) Investigate the contribution of Nef oligomerization to the activation of Hck. The key issue is whether Nef acts in part by bringing two or more molecules of Hck into close proximity, allowing for trans-phosphorylation. 4) Expand the study to other members of the Src kinase family. Of particular interest in this regard is Lck, the kinase activity of which appears to be suppressed by Nef. This Aim will address why interaction of Nef with Lck, which appears to involve both the SH2 and SH3 domains, leads to suppression rather than stimulation of kinase activity. Results of these studies will provide important mechanistic insights to guide the development of agents designed to block Nef-Src kinase interaction in vivo. Such agents may interfere with the actions of Nef, an essential progression factor in AIDS.